1. Field of the Invention
The present invention relates to beam combiners, in general, and to a beam combiner of a specific configuration, in particular.
2. Description of Related Art
Confocal laser microscopy is a tool for the defined addressing of microscopic objects. This method sets very high requirements for the confocal laser microscopy is a tool for the defined addressing of microscopic objects. This method sets very high requirements to the imaging performance of the optic system, which typically is close to the deflection-limited resolution capability. Based on confocal laser scanning microscopy a multitude of methods for examining and influencing microscopic objects has been suggested, such as e.g., Denk in U.S. Pat. No. 5,034,613, TPA, Liu in U.S. Pat. No. 6,159,749, for “Highly Sensitive Bead-Based Multi-Analyte Assay System Using Optical Tweezers”, or Karl Otto Greulich in “Micromanipulation by Light in Biology and Medicine” 1999. The combination of an imaging point and/or line scanning system and a manipulator system form the core of such arrangements. Interest in the observation and analysis of fast microscopic processes creates new devices and methods, e.g., ZEISS line scanner LSM 5 LIVE], with its combination with the above-mentioned manipulation methods leading to new insights. Of particular interest is the simultaneous manipulation and observation of microscopic processes (U.S. Pat. No. 6,094,300 and DE 102004034987A1). Therefore modern microscopes attempt to offer as high a number of flexible coupling and decoupling sites as possible (DE 102004016433A1, for “Tube-Type Revolver With At Least Four Positions For Injecting Or Extracting Light Into Or From A Laser Scanning Microscope”). The simultaneous availability of at least two coupling sites for independent scanning systems is particularly important, here, in order to avoid limitations in the temporal resolution based on slow mechanical switching processes. In addition to the tube interface additional coupling sites are possible at the sides of the support of the microscope (preferably in an expanded infinite space: “Sideports”) as well as at the back of the support (“rearports”) as well as at the bottom (“baseports”).
In principle, arrangements with a common irradiation direction (either top light or passing light) or opposite irradiation directions (top light and passing light) are possible. In addition to an applicative background, frequently the common irradiation direction is preferred for device-technological reasons.
In this case, the use of at least one element is necessary, which combines the incoming beams of both devices in the space between the scanners of the simultaneously operating scan systems and the lens, in order to ensure the greatest system flexibility, in the support connection of the scan modules but also in the wavelengths and the polarization of the combined lasers. Both for the manipulation as well as the imaging system the spectral range of use can generally span from the ultraviolet to the infrared spectrum. Typically applied wavelengths for manipulation are e.g., 351, 355, and 364 nm (photo-uncaging), 405 nm (Photo conversion, Kaede, Dronpa, PA-GFP), 488 and 532 nm (photo bleaching, FRET, FRAP, FLIP) as well as 780-900 nm (multi-photon-bleaching e.g., MPFRAP, 2-photon uncaging, direct multiphoton simulation). Depending on the combined wavelengths as well as the coupling sites of the imaging and manipulating system numerous types of dichroic combiners result for useful applications. FIG. 1 shows graphs of the transmission T for a selection of potential combiner types with the manipulation wavelengths 355 nm, 405 nm, 488 and 532 nm being used both in the transmission and the reflection direction. Neutral combiners (e.g., T20/R80) are here universally used for various applications and additionally allow in a simple manner applications, in which both for the imaging system as well as for the manipulating system the same laser wavelength is used (in particular FRAP). Typically a motorized switching device is provided for the various beam combiner types, such as e.g., a motorized reflector revolver in the area of the infinite space between the lens and the tube lens.
In practical application, numerous requirements must be fulfilled by the described beam combiner element, which are to be considered when the element is designed, and which are not discussed in U.S. Pat. No. 6,677,566 B2.
Typical problems of such combiners are the potential occurrence of interferences with identical incline, the protection of the image quality, and the overlapping of the two beam paths.
First, it must be ensured that the beam combiner element does not interfere with the imaging performance of the laser scanning microscope. For example, the fitting requirements of the beam irradiator element are to be selected such that no astigmatism develops.
Due to the limited reflection coefficient at the front and back of a plane-parallel beam combiner, as a result of the reflections, interferences with the same incline may occur, which lead in the focal level of the microscopic lens to a modulation of the amplitude of the exciting light. Typical consequences on the transmission T are therefore
  T  =      1          1      +              m        ⁢                                  ⁢                              sin            2                    (                      2            ⁢            π            ⁢                                                  ⁢                          d              λ                        ⁢                                                            n                  2                                -                                                      sin                    2                                    ⁢                  α                                                              )                    
as shown in FIGS. 2a and 2b, with the modulation m=4R/(1−R) being connected to the geometric median of the reflection coefficient R2=R1R2, λ representing the wavelength, d the thickness, and n the refractive index of the combiner. α represents the angle, at which the splitter is hit and ranges typically up to 0.055 for the field-of-view number 18. The combining element is located in a beam path range, in which during the scanning process (imaging or sample manipulation) the angle of incidence α constantly changes.
Accordingly, the interferences of identical inclines occurring at the plane-parallel combiner lead to a periodic amplitude modulation of the incoming light intensity in the field-of-vision. In practice, this leads to disturbing lines in the imaging system and/or in a linearly varying manipulation efficiency. The reflectivities R1 and R2 depend both on the wavelength (in particular dichronic combiners) as well as the polarization of the laser radiation shining in from, with the latter also being predetermined by the original polarization of the scanning module and its assembly position at the support. With an increasing wavelength the interference lines occurring in the image become larger and their modulation depth becomes greater. In this dielectric combiner, the direction of polarization primarily influences the effects of the anti-reflective (AR) coating. Due to the fact that the combiner is positioned at 45° in the radiation path, which is near the Brewster-angle for glass, the p-component is naturally reflected to a lesser extent than the s-component. In order to achieve modulation below m=0.04 R=0.01 must be realized, which leads to various partial ratios R1 to R2<0.001. Practically such blooming cannot be achieved by the sometimes large spectral range (cf. FIG. 1) for technical reasons even under p-polarization. In practice, it is therefore not possible to avoid the disturbing interference lines simply by optimizing the anti-reflective coating of the combiner.
A potential solution would be a software-based filtering of the measured signals. Due to the fact that combiners regularly create interference lines, the method of the Fourier-filtering is particularly obvious, here. However, software-based filter methods are known to produce a spatial loss in resolution and thus are not acceptable for the application, here.